Immunosuppressive agent

ABSTRACT

The present invention provides an immunosuppressive agent containing an effective amount of one or more of the macrolide antibiotics of the formula (I): ##STR1## (wherein each of R 1 , R 2 , R 3  and R 4  is a methyl or ethyl group). The immunisuppressive agent has excellent immuno-suppressive activity and can be employed as an agent for suppressing rejection occurring after organ transplantation and as an agent for treating autoimmune diseases.

The present invention was accomplished with the aid of NationalInstitute of Health, National Eye Institute Grant EY03804.

TECHNICAL FIELD

The present invention relates to an immunosuppressive agent containingan effective amount of one or more of the macrolide antibiotics of theformula (I): ##STR2## (wherein each of R₁, R₂, R₃ and R₄ is a methyl orethyl group).

BACKGROUND ART

Macrolide antibiotics of the formula (I) according to the presentinvention are described in, for example, Helvetica Chemica Acta 38,1445-1448 (1955), id. 45, 129-138 and 620-630 (1962), Journal ofAntibiotics 23, 105-106 (1970), id. 24, 347-352 (1971), etc., and theprocess for preparing these antibiotics and their physicochemicalproperties are already known. Further, it is known that these compoundsare effective in extermination of vermin such as acarids and cockroaches(see the specification of Japanese Patent Publication No. 46-28100), andit is also known that said macrolide antibiotics are useful as medicinesfor domestic animals and fowls, more specifically as growth promotingagents for animals (see the specification of Japanese Patent PublicDisclosure No. 54-40178) and anti-coccidiosis agents (see thespecification of Japanese Patent Public Disclosure No. 53-91143).

As immunosuppressive agents, there are known alkylating agents such ascyclophosamide, nucleic acid antimetabolites such as 6-mercaptopurineand azathiopurine, antibiotics such as mitomycin C, steroids, folic acidantagonists such as methotrexate, and plant alkaloids such as colchicineand vinblastine. These immunosuppressive agents are used as agents forsuppressing rejection which may occur after transplantation of humanorgans or as medicines for treating patients suffering from autoimmunediseases. It has recently been noticed that cyclic polypeptides whichare typified by cyclosporin A have immunosuppressive activity andnoticeably suppress rejection occurring after organ transplantation. Atthe same time, vigorous studies have been made to achieve application ofcyclic polypeptides to autoimmune diseases, and utility of thesepolypeptides has been confirmed.

The present inventors made exhaustive studies on the activity ofcompounds of formula (I) on the immune system which have a cyclicstructure similar to that of cyclosporins and, as a result, they foundthe fact that compounds of the formula (I) have immunosuppressiveactivity. The present invention is based on this finding.

DISCLOSURE OF THE INVENTION

Compounds of the formula (I) are produced by cultivating Streotomycesaureus (FERM-P No. 233), and practical examples are the following fivedifferent kinds of compound listed in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Compound No.                                                                  1            2        3        4      5                                       ______________________________________                                        R.sub.1 CH.sub.3 C.sub.2 H.sub.5                                                                        C.sub.2 H.sub.5                                                                      C.sub.2 H.sub.5                                                                      C.sub.2 H.sub.5                       R.sub.2 CH.sub.3 CH.sub.3 C.sub.2 H.sub.5                                                                      C.sub.2 H.sub.5                                                                      C.sub.2 H.sub.5                       R.sub.3 CH.sub.3 CH.sub.3 CH.sub.3                                                                             C.sub.2 H.sub.5                                                                      C.sub.2 H.sub.5                       R.sub.4 CH.sub.3 CH.sub.3 CH.sub.3                                                                             CH.sub.3                                                                             C.sub.2 H.sub.5                       m.p. (°C.)                                                                     148-149  63-64    73-74  79-80  105-106                               ______________________________________                                    

Macrolide antibiotics of the present invention which are obtained byfermentation are mixtures usually containing as principal componentsabout 10% of Compound No. 3, about 40% of Compound No. 4 and about 50%of Compound No. 5, the mixtures being referred to as polynactin complex.

Compounds of the formula (I) are useful as immunosuppressive agents.More specifically, they are employed as agents for suppressing rejectionthat occurs after organ transplantation and as agents for treatingautoimmune diseases. Examples of autoimmune diseases include rheumatoidarthritis, systemic lupus erythematosus (SLE), glomerulonephritis,autoimmune diseases in the ophthalmic region such as uveitis, andautoimmune diseases in the thyroid.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing the results of measurement of the anti-SRBCantibody production suppressing effects of selected compounds of thepresent invention by the PFC method.

BEST MODE FOR CARRYING OUT THE INVENTION

The compounds of the formula (I) may be prepared in the form, forexample, of oral or parenteral administration agents by conventionalmeans of formulation. Preferable forms for oral administration includetablets, capsules, granules and liquid preparations.

The dosage of a compound of the formula (I) to human is generally 10-500mg per day, preferably 50-300 mg per day, although the dosage rangeslightly differs depending on the method of administration. It is alsopossible to use two or more different kinds of compound of the presentinvention in combination.

The compounds of the formula (I) of the present invention show an acuteoral toxicity (LD₅₀) of 2500 mg/kg or more on mice and rats.

The immunosuppressive effects of the compounds of the formula (I) of thepresent invention were tested in terms of (1) the anti-SRBC antibodyproduction suppressing effect both in vitro and in vivo and (2) theeffect against experimental autoimmune uveitis.

EXPERIMENTAL EXAMPLES

(1) Anti-SRBC PFC Test

(a) First, 5×10⁵ sheep red blood cells (SRBC) were added to 2×10⁶ BALB/Cmouse spleen cells suspended in an RPMI-1640 medium (containing 5% offetal bovine serum), and cultivated for 4 days. Then, the number ofcells producing anti-SRBC antibodies was measured by the PFC(plaque-forming cell) method. The results of the measurement are shownin FIG. 1. It should be noted that the compounds of the presentinvention were added after being dissolved in methanol, and methanolalone was added to the control group. "Nos." given to the compounds inthe figure respectively correspond to "Nos." of the compounds shown inTable 1. (The same is the case with Table 2 below).

As will be clear from FIG. 1, all the compounds of the present inventionshowed strong PFC production suppressing effects.

(b) First, 5×10⁸ SRBCs were administered intravenously to BALB/C mice(male, 6-week old) for primary sensitization. Then, Compound No. 5 ofthe present invention, which was suspended in olive oil, wasintraperitoneally administered to the mice at the following threedifferent times, that is, immediately after the primary sensitization,24 hours thereafter, and 48 hours thereafter. Four days after theprimary sensitization, spleen cells of the mice were extracted to formsingle-cell suspensions, and the number of cells producing anti-SRBCantibodies was measured by the PFC method. Olive oil alone wasadministered to the control group. The results of the measurement areshown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                                   dosage    Number of                                                Compound No.                                                                             (mg/kg)   animals    PFC/spleen                                    ______________________________________                                        Olive oil  --        12         290000 ± 15200                             Compound No. 5                                                                           10        6          228000 ± 26000.sup.a                                  50        6          176000 ± 17000.sup.b                       ______________________________________                                         .sup.a P < 0.05,                                                              .sup.b P < 0.001                                                         

As will be clear from Table 2, the compounds of the present inventionsignificantly suppressed PFC production in mouse spleen cells.

(2) Effect against Experimental Autoimmune Uveitis (EAU)

(a) Effect on Development of Uveitis

(Method of Experiment)

Experimental animals: Lewis rats (female) each having a weight of about170 g

Immunization: S antigen extracted and purified from the bovine retinawas emulsified in an equivalent of complete Freund's adjuvant (a productavailable from DIFCO), and 50 μg of this emulsion was injected into theplanta pedis of each of the rats.

Tested compounds: Compounds Nos. 3, 4 and 5 shown in Table 1 were mixedtogether at a ratio of 1:4:5, and the mixture (hereinafter referred toas "PN") was suspended in olive oil using a glass homogenizer to preparePN samples having various concentrations, which were then injected tothe rats. Olive oil alone was injected into rats in the control group.

Administration method: For three groups of rats, 10 mg of PN (in 0.1 mlof olive oil), 20 mg of PN (in 0.2 ml of olive oil) and 30 mg of PN (in0.3 ml of olive oil) were injected into the femoral muscles of rats,respectively, once a day from the seventh day to the fourteenth dayafter the immunization. For one group of rats, 10 mg of PN (in 0.1 ml ofolive oil) was injected into the femoral muscle of each of the rats oncea day from the day of the immunization to the fourteenth day thereafter.

Observation: Development of uveitis was examined by observing theanterior portion of the eye with a slit lamp.

(Results)

Results of observation with a slit lamp are shown in Table 3. As will beclear from Table 3, acute uveitis occurred in 10 to 11 out of 12 eyes ofthe rats in the control group (injected with S antigen and having no PNadministered thereto) at the fifteenth day after the sensitization. Forthe groups of rats having PN administered thereto from the seventh dayafter the sensitization, acute uveitis occurred in 4 out of 10 eyes ofthe rats at a dosage of 10 mg of PN per day, in 6 out of 16 eyes of therats at a dosage of 20 mg per day, and in only one out of 14 eyes of therats at a dosage of 30 mg per day. Acute uveitis did not occur in thegroup of rats to which 10 mg of PN per day had been administered fromthe day of the sensitization.

                  TABLE 3                                                         ______________________________________                                        PN                    Incidence of disease                                    injection             Control   PN-administered                               (mg/rat/day)                                                                           Injection period                                                                           group     groups                                        ______________________________________                                        10       7th day-14th day                                                                           11/12     4/10                                          20       7th day-14th day                                                                           10/12     6/16                                          30       7th day-14th day                                                                           10/12     1/14                                          10       0-14th day   10/12     0/8                                           ______________________________________                                    

Accordingly, it has been confirmed that the compounds of the presentinvention are significantly effective against experimental autoimmuneuveitis.

(b) Effect on Dermal Test by Secondary Immune Response

(Method of Experiment)

Rats were primarily sensitized by the immunization method described in(a). After the sensitization, olive oil alone was injected into thefemoral muscles of rats in two control groups, while 20 mg of PN (in 0.2ml of olive oil) was injected into the femoral muscles of rats in onegroup once a day from the day of the immunization to the fourteenth daythereafter and also injected into the femoral muscles of rats in anothergroup once a day from the seventh day to the fourteenth day after theimmunization. On the fifteenth day after the sensitization, bovine Santigen (100 μg/0.1 ml) was subcutaneously injected into the ventralportion of each of the rats so that they were secondarily sensitized,and the thickness of the skin of each rat was measured with a caliper ateach of the times, that is, 3 hours and 24 hours after the secondarysensitization. Two different kinds of control group were prepared forcomparison, that is, a control group that was secondarily sensitized (+Santigen) and another control group which was subjected to no secondarysensitization (-S antigen).

(Results)

The results of the skin test are shown in Table 4. As will be understoodfrom Table 4, although there is no significant difference between thecontrol group (+S antigen) and the PN-administered groups in the testcarried out when 3 hours had elapsed after the secondary sensitization,the PN-administered groups show lower values than those of the controlgroup (+S antigen) in the test carried out when 24 hours had elapsedafter the secondary sensitization.

                  TABLE 4                                                         ______________________________________                                        Control groups    PN-administered                                             -S           +S       (20 mg/rat/day) groups                                  antigen      antigen  7th day-14th day                                                                           0-14th day                                 ______________________________________                                        3 hours 3.0 mm   9.0 mm     9.0 mm     9.0 mm                                 after            8.0      8.0        8.5                                      secondary        9.0      8.0        9.5                                      sensiti-                                                                      zation                                                                        24 hours                                                                              3.0 mm   9.0 mm     3.0 mm     7.0 mm                                 after            9.0      6.0        5.0                                      secondary        8.5      6.0        5.5                                      sensiti-                                                                      zation                                                                        ______________________________________                                    

Accordingly, it has been confirmed that the compounds of the presentinvention have an immunosuppressive effect.

FORMULATION EXAMPLE

Sorbitol is dissolved in purified water in an amount which is a quarterof the necessary amount according to the following prescription example.This solution is stirred at a high speed using Polytron® (a productavailable from KINEMATICA) and while doing so, Compound No. 5 shown inTable 1 and sodium carboxymethyl cellulose are gradually added to thesolution and homogenized. Thereafter, the remaining portion of thepurified water is added to prepare a suspension. The prepared solutionis pipetted into 5-ml brown ampules, sealed and autoclaved at 115° C.for 30 minutes to prepare an injection.

PRESCRIPTION EXAMPLE

    ______________________________________                                                        Concentration (w/v%)                                          ______________________________________                                        Compound No. 5    1                                                           Sorbitol          5                                                           Sodium carboxymethyl cellulose                                                                  2                                                           Purified water    q.s                                                         Total amount      100                                                         ______________________________________                                    

INDUSTRIAL APPLICABILITY

As has been described above, the compounds of formula (I) of the presentinvention are useful as immunosuppressive agents and can be employed asagents for suppressing rejection occurring after organ transplantationand as agents for treating autoimmune diseases.

What is claimed is:
 1. A method of treating a patient having anautoimmune disease selected from the group consisting of rheumatoidarthritis, systemic lupus erythematosus, glomerulonephritis, autoimmuneuveitis, and autoimmune disease in the thyroid, comprising administeringto said patient, in order to suppress anti-SBRC antibody production,from about 10 to about 500 mg per day of an antibiotic of the followingformula: ##STR3## wherein each of R₁, R₂, R₃, and R₄ is selected fromthe group consisting of methyl and ethyl, in a pharmaceuticallyacceptable carrier.
 2. The method of claim 1 wherein the autoimmunedisease is autoimmune uveitis.
 3. A method of suppressing anti-SBRCantibody production in a patient comprising administering to saidpatient from about 10 to about 500 mg per day of an antibiotic of thefollowing formula: ##STR4## wherein each of R₁, R₂, R₃, and R₄ isselected from the group consisting of methyl and ethyl, in apharmaceutically acceptable carrier.
 4. The method of claim 3 whereinthe antibiotic is administered in an amount of from about 50 to about300 mg per day.